xcex2-cells are specialized cells that secrete insulin and are found in pancreatic islets. Insulin belongs to a group of protein/polypeptide hormones. Insulin increases the rate of synthesis of glycogen, fatty acids, and proteins and stimulates glycolysis and cell proliferation. It also promotes the transport of glucose, and some other sugars, and amino acids into muscle and fat cells. Insulin levels are regulated to maintain glycemic homeostasis, and an important mechanism for regulating insulin production, and hence insulin levels, is xcex2-cell mass.
During the lifetime of an individual metabolic needs can change drastically, requiring dynamic changes in cells and tissues that regulate homeostasis. During pregnancy (Marynissen et al., Diabetes 36:883-891, 1987) xcex2-cell mass increases, as well as in response to obesity (Kloppel et al., Surv. Synth. Pathol. Res. 4:110-125, 1985). These increases in xcex2-cell mass are attributed to an increased requirement for insulin to maintain normal glucose levels (Parsons et al., Endocrinology 130:1459-1466, 1992). It has also been shown that xcex2-cell mass normally decreases post-partum, primarily by apoptosis (Scaglia et al., Endocrinology 136:5461-5468, 1995).
It is generally believed that increases in xcex2-cell mass occurs in three ways: 1) an increase in cell size and function; 2) increased proliferation of mature xcex2-cells; and/or 3) increased,recruitment and differentiation of xcex2-cell progenitors. In diabetic mice, animals that received islet transplants and then achieved normal glycemia, showed xcex2-cell hypertrophy, rather than an increase in cell replication (Montana et al., J. Clin. Invest. 91:780-787, 1993). Adult xcex2-cell regeneration has been demonstrated in rodents (Hellerstrom et al., in xe2x80x9cThe Pathology of the Endocrine Pancreas in Diabetesxe2x80x9d, P. J. Lefebvre and D. G. Pipeleers, eds., pp. 141-170, Springer-Verlag, Heidelberg, 1988). In partially pancreatectomized rats both preexisting xcex2-cells, as well as proliferation and differentiation of precursor cells, have been demonstrated to expand (Bonner-Weir, Diabetes Nutr. Res. 5, Supp. 1:21-25, 1992).
Several factors have been shown to increase xcex2-cell mass. These factors include glucose (Woerner, Anal. Rev. 71:33-57, 1938), IGF-I (Rabinovitch et al., Diabetes 31:160-164, 1982), reg protein (Terazono et al., J. Biol. Chem. 263:2111, 1988) and possibly a combination of TGF-xcex1 and gastrin (Bonner-Weir, Recent Prog. Hormone Res. 12:91-104, 1994). While some factors have been shown to increase xcex2-cell mass in vitro or in vivo, understanding of the process is poorly understood and the possibility that other unidentified factors are involved is likely.
Recently a new member of the insulin superfamily has been identified, early placenta insulin-like factor or placentin (Chassin et al., Genomics 29:465-470, 1995). Placentin cDNA was isolated from first trimester human placenta and found to have a 139-amino acid open reading frame. Based on homology to the rest of the insulin superfamily it was predicted that placentin, like preprorelaxin and preproinsulin, would have a signal sequence, followed by the B chain, C peptide, A chain. The mature molecule would have the signal peptide and C peptide removed, with the B and A chains joined by both inter- and intra-chain disulfide bonds (Chassin et al., 1995, ibid. and James et al., Nature 267:544-546, 1977). The B-chain, C-peptide, A-chain motif is found in several other proteins, including relaxin (U.S. Pat. No. 4,835,251), insulin-like growth factors (IGF) I and II (Bang and Hall, in xe2x80x9cInsulin-like Growth Factorsxe2x80x9d, P. N. Schofield (ed.), pp. 151-177, Oxford University Press, Oxford, 1992), and Leydig Factor (Bullesbach et al., J. Biol. Chem. 270:16011-16015, 1995). Unlike other members of the insulin superfamily, IGF I and IGF II have D and E domains that are cleaved post-translationally. Cysteines that are involved in disulfide bonds are conserved in.all the members of the family and play a role in the tertiary structure of the molecules.
Placentin has been shown to stimulate 3H-thymidine uptake in human placental 3AsubE cells and stimulate human chorionic gonadotropin production in primary cultures of trophoblasts (Koman et al., J. Biol. Chem. 271:20238-20241, 1996). This activity suggests that placentin may play a role during placental development. However, the present inventors, surprisingly, have found that a molecule encoded by the DNA for placentin, but a different amino acid structure, increases xcex2-cell mass and may be useful in treatment of diabetes, and further that the biologically active molecule differs from the molecule described in the art.
The present invention provides proteins produced by a method comprising: culturing a host cells into which has been introduced a DNA expression vector comprising a transcription promoter; a DNA segment comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 76 to nucleotide 417; and a transcription terminator, wherein said host cell expresses the polypeptide encoded by said DNA segment and recovering said protein.
In another embodiment, the host is a mammalian cell. In another embodiment, the host has had a second DNA expression vector introduced into it, wherein the second expression vector comprises a transcription promoter; a DNA segment encoding an endoprotease; and a transcription terminator, wherein said host cell expresses the a DNA segment comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 76 to nucleotide 417 and said DNA segment encoded by the endoprotease.
In another aspect, the present invention provides an isolated and purified protein comprising a first polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 26 (Ala) to residue 110 (Ser) or 114 (Arg); and a second polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 115 (Ser) to residue 139 (Thr), wherein said first polypeptide and said second polypeptide are capable of disulfide associating.
In another aspect, the present invention provides an isolated and purified protein comprising a first polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 26 (Ala) to residue 48 (Lys), 49 (Thr) or 50 (Phe); and a second polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 115 (Ser) to residue 139 (Thr), wherein said first polypeptide and said second polypeptide are capable of disulfide associating.
In another aspect, the present invention provides a method of stimulating proliferation of pancreatic islet comprising administering to a mammal in need thereof, an amount of an isolated and purified polypeptide comprising: a first polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 26 (Ala) to residue 110 (Ser) or 114 (Arg); and a second polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from amino acid residue 115 (Ser) to residue 139 (Thr), and wherein said first polypeptide and said second polypeptide are capable of disulfide associating, sufficient to produce a clinically significant increase in insulin secretory capacity.
In another aspect, the present invention provides a method of stimulating proliferation of pancreatic islets comprising administering to a mammal in need thereof, an amount of an isolated and purified polypeptide comprising: a first polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 26 (Ala) to residue 48 (Lys), 49 (Thr) or 50 (Phe); and a second polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from amino acid residue 115 (Ser) to residue 139 (Thr), and wherein said first polypeptide and said second polypeptide are capable of disulfide associating, sufficient to produce a clinically significant increase in insulin secretory capacity.
In other embodiments, the present invention provide methods wherein the clinically significant increase in insulin secretory capacity results in a decrease in fasting plasma glucose levels.
In other embodiments, the present invention provide methods wherein the isolated and purified protein is administered in combination with an insulin sensitizer.
In another aspect, the present invention provides a method for stimulating in vitro proliferation of pancreatic islet cells comprising culturing islets with an amount of an isolated and purified protein comprising: a first polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 26 (Ala) to residue 110 (Ser) or 114 (Arg); and a second polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from amino acid residue 115 (Ser) to residue 139 (Thr), and wherein said first polypeptide and said second polypeptide are capable of disulfide associating, sufficient to produce an increase in the number of islet cells as compared to islet cells cultured in the absence of the protein.
In another aspect, the present invention provides a method for stimulating in vitro proliferation of pancreatic islet cells comprising culturing islets with an amount of an. isolated and purified protein comprising: a first polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 26 (Ala) to residue 26 (Ala) to residue 48 (Lys), 49 (Thr) or 50 (Phe); and a second polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from amino acid residue 115 (Ser) to residue 139 (Thr), and wherein said first polypeptide and said second polypeptide are capable of disulfide associating, sufficient to produce an increase in the snumber of islet cells as compared to islet cells cultured in the absence of the protein.
In other embodiments, the present invention provides methods wherein said cells are cultured in 0.1 ng/ml to 100 ng/ml of said protein.